Our long term goal is to obtain an understanding of the role in smooth muscle of bound inositol-(1,4,5)trisphosphate (ins(1,4,5)P3). In experiments proposed in this application, we ask questions about the site and the characteristics of binding of ins(l,4,5)P3 in swine trachealis smooth muscle (STSM), and how bound Ins(1,4,5)P3 may be released during development of force. We were led to this study by previous results which indicated that most Ins(l,4,5)P3 in unstimulated STSM cells is bound, or compartmentalized, and that bound or compartmentalized ins(l,4,5)P3 may be released during muscarinic-receptor activation of muscle. We will study cytosolic proteins and proteins which are bound to the cytoskeleton, or contractile apparatus. We will start with studies of Ins(l,4,5)P3 binding to aldolase because preliminary data indicate ins(l,4,5)P3 is bound to this compound. We will also determine the characteristics of Ins(l,4,5)P3 binding to non-aldolase- cytosolic proteins and proteins released from the cytoskeleton or contractile apparatus. We will utilize 4 different preparations: (i) intact STSM; (ii) a myofilament-cytoskeleton preparation which binds 50 -75% of cellular aldolase and a significant fraction of total bound ins(l,4,5)P3; (iii) a purified smooth muscle aldolase preparation; (iv) a synthetic thin filament preparation. The major hypothesis tested is that during muscarinic activation of STSM, Ins(1,4,5)P3 bound to aldolase in the cytosol, and/or to aldolase complexed with contractile proteins, is released providing free ins(l,4,5)P3 which drives calcium release from the sarcoplasmic reticulum; that Ins(l,4,5)P3 release from its bound site on aldolase is triggered by aldolase substrate or ligands which bind to aldolase.